Biotechnology in the 21st century presents the possibility of new solutions to food, environment and health problems, with the ultimate object of promoting human prosperity. In recent years, the technology of using stem cells has been considered as a new way to treat incurable diseases. Formerly, organ transplantation, gene therapy, etc., were proposed for the treatment of incurable human diseases, but their use has not been achieved efficiently due to immunorejection, a small supply of organs, and insufficient knowledge of genes.
For this reason, with increasing interest in stem cell research, it has been recognized that totipotent stem cells having the ability to form all organs by proliferation and differentiation can not only treat most diseases but also fundamentally heal organ injuries. Also, many scientists have suggested the applicability of stem cells for the regeneration of all the organs and the treatment of incurable diseases, including Parkinson's disease, various cancers, diabetes and spinal damage.
Stem cells refer to cells having not only self-replicating ability but also an ability to differentiate into at least two types of cells, and can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells (MSCs).
Totipotent stem cells are cells having totipotent properties capable of developing into one perfect individual, and these properties are possessed by cells up to the 8-cell stage after the fertilization of an oocyte and a sperm. When these cells are isolated and transplanted into the uterus, they can develop into one perfect individual. Pluripotent stem cells, which are cells capable of developing into various cells and tissues derived from the ectodermal, mesodermal and endodermal layers, are derived from an inner cell mass located inside of blastocysts generated 4-5 days after fertilization. These cells are also called embryonic stem cells and can differentiate into various other tissue cells but cannot form new living organisms.
Multipotent stem cells were first isolated from adult bone marrow (Y. Jiang et al., Nature, 418: 41, 2002), and then also found in other various adult tissues (C. M. Verfaillie, Trends Cell Biol., 12: 502, 2002). In other words, although the bone marrow is the most widely known source of stem cells, the multipotent stem cells were also found in the skin, blood vessels, muscles and brains (J. G. Tomas et al., Nat. Cell Biol., 3: 778, 2001; M. Sampaolesi et al., Science, 301: 487, 2003; Y. Jiang et al., Exp. Hematol., 30: 896, 2002). However, stem cells in adult tissues, such as the bone marrow, are very rarely present, and such cells are difficult to culture without inducing differentiation, and thus difficult to culture in the absence of specifically screened media.
The reason why it is important to establish cell lines of such multipotent stem cells is because of the objectives of the research on the proliferation, lyophilization and characterization of stem cell lines, drug tests, and the autologous, allogeneic and xenogeneic transplantation of stem cell lines.
Also, animal models have been of particular importance in regenerative medicine for the repair or restoration of function of injured or damaged tissues. Over 370 genetic diseases have been found in canines and majority of these canine diseases resemble human diseases and dysfunctions. Thus, canines are of increasing importance as animal models for the research of the mechanism and pathogenesis of human genetic diseases, particularly X-linked severe combined immunodeficiency and combined genetic rare recessive diseases such as Duchenne muscular dystrophy, which are difficult to study directly in humans. Such canines are useful animal models for studying not only solid organ transplantation, but also for studying the pathogenic mechanisms of human diseases, including prostate cancer, cardiovascular diseases, bone regeneration, diabetes, leukemia and spinal cord injury, and for testing new therapeutic methods. In addition, canines are ideal large animal models for studying various therapeutic methods such as stem cell transplantation and gene therapies.
Therefore, isolation and characterization of stem cells derived from various canine tissues have become important issues in the stem cell field. Conventionally, there have been studies on stem cells from human and mouse tissues in various fields. As described above, canine aminals are useful large animal models for studying human diseases, but studies on canines have not yet been sufficient. Canine stem cells can be isolated from adipose tissue, bone marrow, umbilical cord blood and the like, but these are obtained in limited amounts, and the method of obtaining adipose tissue or bone marrow from individuals is invasive and causes pain.
In the current state of technology, in order to use stem cells as cell therapeutic agents, it is required to standardize the culture conditions under which an undifferentiated state can be maintained. In addition, because stem cells isolated from tissues are present as a mixture of various kinds of cells, it is required to develop technology capable of culturing homogeneous stem cells on a mass scale. In particular, methods for isolating stem cells from tissues or blood generally include, for example, cell sorting utilizing antibodies for a number of surface antigens. However, this method has a shortcoming in that the surface antigens of stem cells should be known. In addition, a common surface antigen (hereinafter referred to as “marker”) for stem cells is not yet known. Also, various markers for stem cells have not been developed, and known markers for stem cells are expressed at different levels depending on the differentiation state of stem cells. Particularly, a system of sorting cells according to the expression level of the markers is expensive. Due to such shortcomings, the use of the cell sorting method has been greatly limited.
The placenta plays an important role in the development and survival of a fetus by supplying nutrients and oxygen thereto. Generally, the placenta is disposed of as medical waste after delivery. However, recent studies indicate that human amniotic tissue is a source rich in stem cells, and many studies on stem cells derived therefrom have been conducted. In clinical applications, amniotic tissue has effects on wound healing and retinal reconstitution. The amnion may possibly contain stem cells in a mixture with other monocytes and other stem cells. Under culture conditions for such mixed cells, the distribution of nutrients cannot be uniform, thereby causing heterogeneity in differentiation of cells. Conclusively, the problem that the cells cannot be produced as a homogeneous cell population serves as a fatal disadvantage, as when they are used as the therapeutic agent the actual effect may be different from the intended effect. Therefore, there is an urgent need for the development of effective culture technology that makes it possible to obtain homogeneous adult stem cells in a large quantity.